Compositions and methods to control angiogenesis with cupredoxins

ABSTRACT

The present invention relates to compositions comprising cupredoxins, and their use to inhibit angiogenesis in mammalian cells, tissues, and animals, and particularly the angiogenesis that accompanies tumor development and particularly in humans. Specifically, the present invention relates to compositions comprising the cupredoxin(s), and or peptides that are variants, derivatives or structural equivalents of cupredoxins, which retain the ability to inhibit angiogenesis in mammalian cells, tissues or animals. These compositions may be peptides or pharmaceutical compositions, among others. The compositions of the invention may be used to treat any pathological condition that has as a symptom or cause, inappropriate angiogenesis, and particularly inappropriate angiogenesis related to tumor development.

RELATED APPLICATIONS

The present application claims the benefit of priority under 35 U.S.C.§§119 and 120 to and is a continuation of U.S. patent application Ser.No. 12/477,358, filed Jun. 3, 2009, issued as U.S. Pat. No. 8,124,055 onFeb. 28, 2012, which is a continuation of U.S. patent application Ser.No. 11/488,693, filed Jul. 19, 2006, issued as U.S. Pat. No. 7,556,810on Jul. 7, 2009, which claims priority to U.S. Provisional Patent App.Ser. No. 60/700,297, filed Jul. 19, 2005, and which is acontinuation-in-part of U.S. patent application Ser. No. 11/436,592filed May 19, 2006, issued as U.S. Pat. No. 7,381,701 on Jun. 3, 2008,which claims priority to U.S. Provisional Patent App. Ser. No.60/764,749, filed Feb. 3, 2006, and is a continuation-in-part of U.S.patent application Ser. No. 11/244,105, filed Oct. 6, 2005, issued asU.S. Pat. No. 7,691,383 on Apr. 6, 2010, which claims priority to U.S.Provisional Patent App. Ser. No. 60/616,782, filed Oct. 7, 2004, andU.S. Provisional Patent App. Ser. No. 60/680,500, filed May 13, 2005,and which is a continuation-in-part of U.S. patent application Ser. No.10/720,603, filed Nov. 24, 2003, issued as U.S. Pat. No. 7,491,394 onFeb. 17, 2009, which claims priority to U.S. Provisional Patent App.Ser. No. 60/414,550, filed Aug. 15, 2003, and which is acontinuation-in-part of U.S. patent application Ser. No. 10/047,710,filed Jan. 15, 2002, issued as U.S. Pat. No. 7,084,105 on Aug. 1, 2006,which claims priority to U.S. Provisional Patent App. Ser. No.60/269,133, filed Feb. 15, 2001. The entire content of these priorapplications is fully incorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under research grantsfrom the National Institutes of Health (NIH), Bethesda, Md., U.S.A.,(Grant Numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 andN01-CM97567). The government has certain rights in the invention.

This application contains a Sequence Listing text file electronicallyfiled via the EFS system, which contains a sequence listing. Thematerials recorded in text file are incorporated herein by reference intheir entirety. The text file contains a single file named“14PR.14736txt” (25 KB, created on May 20, 2009).

FIELD OF THE INVENTION

The present invention relates to cupredoxins and variants, derivativesand structural equivalents of cupredoxins, specifically Pseudomonasaeruginosa azurin, and their use in inhibiting angiogenesis in mammals,to treat conditions related to inappropriate angiogenesis in mammals,and in particular in inhibiting angiogenesis associated with tumordevelopment. The invention also relates to pharmaceutical compositionscomprising cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that can be administered to a mammalianpatient, and specifically administered to inhibit angiogenesis.

BACKGROUND

Angiogenesis is the formation of new blood vessels from preexistingendothelial vasculature. Folkman, et al., J. Exp. Med. 133:275-288,(1971). Most tumors require angiogenesis to sustain growth beyond acritical volume of 1-2 mm, when the supply of nutrients and metabolitesbecomes insufficient due to the limits of diffusional exchange. Folkman,J. Nat. Cancer Inst. 82:4-6 (1990). Tumors deprived of angiogenesisremain dormant indefinitely, only to rapidly grow when a blood supply isacquired. Brem et al., Cancer Res. 36:2807-2812 (1976). The degree ofangiogenesis often increases with tumor progression. Dome et al., J.Pathol. 197:355-362 (2002). Further, invasion and metastatic spread oftumors are also thought to be angiogenesis-dependant events. Folkman,Ann Surg. 175:409-416 (1972). The newly formed blood vessels provide aroute for cancer cells to enter the circulatory system and spread todistant parts of the body. Fidler and Ellis, Cell 79:185-188 (1994).

Because angiogenesis is an integral process in the growth and spread oftumors, it is an important focus of cancer therapy. Anti-angiogenesistherapy is effective not only for solid tumors, but also hematopoietictumors, leukemia and myeloma, Bellamy et al., Cancer Res. 59:728-733(1999); Rajkumar et al., Leukemia. 13:469-472 (1999). Endothelial cellsare thought to be better targets for therapy than tumor cells becausethey have a longer generation time and more genetic stability that tumorcells. Endothelial cells are therefore less likely to “escape” therapyby developing drug resistance to the therapy administered.Boehn-Vaiswanathan, Curr. Opin. Oncol. 12:89-94 (2000).

Other conditions suffered by mammals are also related to inappropriateangiogenesis. Wet macular degeneration occurs when blood capillariesinappropriately grow into the retina. Inappropriate angiogenesis hasalso been implicated as a fundamental characteristic of diabeticretinopathy, psoriasis and rheumatoid arthritis, among other diseases.Bussolino et al., Trends Biochem. Sci. 22:251-256 (1997); Folkman, Nat.Med. 1: 27-31 (1995).

What is needed are additional therapies for inappropriate angiogenesis,particularly that which occurs during tumor formation. Such therapiesmay be useful in many conditions that exhibit inappropriate or unwantedformation of new blood vessels.

SUMMARY OF THE INVENTION

The present invention relates to compositions comprising cupredoxins,and their use to inhibit angiogenesis in mammalian cells, tissues, andanimals, and particularly the angiogenesis that accompanies tumordevelopment and particularly in humans. Specifically, the presentinvention relates to compositions comprising the cupredoxin(s), and orpeptides that are variants, derivatives or structural equivalents ofcupredoxins, which retain the ability to inhibit angiogenesis inmammalian cells, tissues or animals. These compositions may be peptidesor pharmaceutical compositions, among others. The compositions of theinvention may be used to treat any pathological condition that has as asymptom or cause, inappropriate angiogenesis, and particularlyinappropriate angiogenesis related to tumor development.

One aspect of the invention is an isolated peptide that is a variant,derivative or structural equivalent of a cupredoxin and that can inhibitangiogenesis in mammalian cells. The cupredoxin may be azurin,pseudoazurin, plastocyanin, rusticyanin, Laz and auracyanin, andspecifically azurin. The cupredoxin may be from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseriagonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa and Vibrio parahaemolyticus, and specifically Pseudomonasaeruginosa. The isolated peptide may be part of a SEQ ID NOS: 1, 3-19,or be a sequence to which SEQ ID NOS: 1, 3-19 has at least 80% aminoacid sequence identity.

The isolated peptide may be a truncation of cupredoxin. In these cases,the isolated peptide may be more than about 10 residues and not morethan about 100 residues. The isolated peptide comprises Pseudomonasaeruginosa azurin residues 50-77, residues 50-67 or residues 36-88 orSEQ ID NOS: 20-24. Further, the isolated peptide may consist ofPseudomonas aeruginosa residues 50-77, residues 50-67 or residues 36-88or SEQ ID NOS: 20-24. Finally, the isolated peptide may compriseequivalent residues of Pseudomonas aeruginosa azurin residues 50-77,residues 50-67 or residues 36-88

Another aspect of the invention is a pharmaceutical composition whichcomprises at least one cupredoxin or isolated peptide in apharmaceutically acceptable carrier. This pharmaceutical composition maycomprise at least two of the cupredoxins or isolated peptides. Further,the pharmaceutical composition may be formulated for intravenousadministration. In some embodiments, the cupredoxin is from Pseudomonasaeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseriagonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa or Vibrio parahaemolyticus, and specifically from Pseudomonasaeruginosa. The cupredoxin may be SEQ ID NOS: 1, 3-19.

Another aspect of the invention are methods to treat a mammalian patientsuffering from a condition related to inappropriate angiogenesis whichcomprises administering to the patient a therapeutically effectiveamount of the pharmaceutical composition. In some embodiments thepatient is human. The patient may be suffering from cancer, and inparticular melanoma, breast, pancreas, glioblastoma, astrocytoma, orlung cancer. In other embodiments, the patient may be suffering from acondition selected from the group consisting of macular degeneration,diabetic retinopathy, psoriasis or rheumatoid arthritis. In thesemethods, the pharmaceutical composition is administered by intravenousinjection, intramuscular injection, subcutaneous injection, inhalation,topical administration, transdermal patch, suppository, vitreousinjection or oral, and specifically by intravenous injection.

In some embodiments of the methods, the pharmaceutical composition isco-administered with at least one other anti-cancer drug, andspecifically at about the same time as another anti-cancer drug. Inother embodiments of the method, the pharmaceutical composition isco-administered with an anti-macular degeneration drug, an anti-diabeticretinopathy drug, an anti-psoriasis drug or an anti-rheumatoid arthritisdrug.

Another aspect of the invention is a kit comprising the pharmaceuticalcomposition in a vial. This kit may be designed for intravenousadministration.

Another aspect of the invention is a method to study angiogenesis or acondition related to inappropriate angiogenesis, comprising contactingthe mammalian cells capable of angiogenesis with a cupredoxin orvariant, derivative or structural equivalent of a cupredoxin andmeasuring the extend of angiogenesis. In some embodiments, the cells arehuman cells. In other embodiments, the mammalian cells are HumanUmbilical Vascular Endothelium Cells (HUVECs).

Another aspect of the invention is an expression vector which encodes avariant, derivative or structural equivalent of a cupredoxin.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa.

SEQ ID NO: 2. Amino acid sequence of P28, Pseudomonas aeruginosa azurinresidues 50-77.

SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 4. Amino acid sequence of rusticyanin from Thiobacillusferrooxidans.

SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobactercycloclastes.

SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis.

SEQ ID NO: 7. Amino acid sequence of azurin from Achromobacterxylosoxidans ssp. denitrificans I.

SEQ ID NO: 8. Amino acid sequence of azurin from Bordetellabronchiseptica.

SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J.

SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidisZ2491.

SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonasfluorescen.

SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonaschlororaphis.

SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa9a5c.

SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus.

SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexusaurantiacus.

SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexusaurantiacus.

SEQ ID NO: 17. Amino acid sequence of cucumber basic protein fromCucumis sativus.

SEQ ID NO: 18. Amino acid sequence of Laz from Neisseria gonorrhoeaeF62.

SEQ ID NO: 19. Amino acid sequence of the azurin from Vibrioparahaemolyticus.

SEQ ID NO: 20. Amino acid sequence of amino acids 57 to 89 of auracyaninB of Chloroflexus aurantiacus.

SEQ ID NO: 21. Amino acid sequence of amino acids 51-77 of Pseudomonassyringae azurin.

SEQ ID NO: 22. Amino acid sequence of amino acids 89-115 of Neisseriameningitidis Laz.

SEQ ID NO: 23. Amino acid sequence of amino acids 52-78 of Vibrioparahaemolyticus azurin.

SEQ ID NO: 24. Amino acid sequence of amino acids 51-77 of Bordetellabronchiseptica azurin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts confocal microscopy images of malignant and normal cellsincubated with P28 labeled with Alexafluor® 568 and the cells are thenstained with DAPI. The indicated cell lines were incubated in theabsence (negative control) or presence (P28) of 20 μM Alexafluor® 568labeled P28 for 2 h at 37° C. The images are indicative of amount ofcellular entry observed. FIG. 1A depicts the Alexafluor® 568fluorescence and control fluorescence of human melanoma, pancreatic,breast (BCA-1), breast (MCF-7), glioblastoma, astrocytoma, lung andprostrate cancer cells. FIG. 1B depicts the Alexafluor® 568 fluorescenceand control fluorescence of human normal fibroblast, pancreas and breastcells. FIG. 1C depicts the Alexafluor® 568 fluorescence and controlfluorescence of human umbilical vein endothelial cells (HUVEC).

FIG. 2 depicts the capillary tube formation by HUVEC cells plated onMatrigel® in the presence or absence of P28. Culture media contained 20ng/ml VEGF. FIG. 2A shows images of HUVEC cells incubated for 4 h at 37°C. with 0.10 μM, 0.30 μM, 0.92 μM, 2.77 μM, 8.33 μM, 25 μM and 75 μM ofP28, and then stained with calcein AM and visualized using fluorescencemicroscopy. In FIG. 2B, the graph shows the average number of tubesformed in peptide treated and control (untreated) cells.

FIG. 3 depicts the results of the scratch wound HUVEC migration assay.In FIGS. 3A-C show the fixed cells that were stained for F-actin andnuclei. In FIG. 3A, HUVEC cells at 90% confluence were scratched using a1 ml plastic pipette tip. In FIG. 3B, the HUVEC cells were scratched andthen incubated in the culture media containing 20 ng/ml VEGF for 24 h at37° C. in the absence of P28. In FIG. 3C, the HUVEC cells were scratchedand then incubated for 24 h at 37° C. in the presence of 25 μM P28. Theinsets of FIGS. 3A-C show the cell density in the area away from thescored area. In FIG. 3D, a bar graph indicates the average # of cells in20 different fields (20∴) of the scratched area in control and P28treated wells (FIGS. 3B and C). Data represent mean±SEM. * indicates thedifferences are statistically significant.

FIG. 4 depicts the images of the localization of cell structuralproteins with and without P28 treatment. HUVEC cells were plated onMatrigel®-coated cover slips, incubated in the culture media containing20 ng/ml VEGF in the presence or absence of P28 peptide (25 μM) for 4and 24 h, fixed, and processed for staining of CD31/PECAM-1, paxillin,Fak (focal adhesion kinase), vinculin, WASP (Wiskott Aldrich Syndromeprotein) and β-catenin. Each figure pertains to the detection ofparticular structural protein: FIG. 4A is CD31/PECAM-1; FIG. 4B ispaxillin; FIG. 4C is Fak; FIG. 4D is WASP; FIG. 4E is vinculin; and FIG.4F is β-catenin. Each figure is divided into four panes which show theimage of the localization of the fluorescent markers used. Each pane isnumbered to indicate the fluorescent marker detected: 1=F-actin; 2=DAPI;3=FITC-Protein of interest; 4=merged image. Arrows indicate thelocalization of the protein of interest.

FIG. 5 depicts Mel-2 cells which were treated with increasingconcentrations of P28 for 24, 48, and 72 hours. The number of cells intreated and control wells were counted using a Coulter counter. Datarepresent percentage of cell growth inhibition when compared to controlcultures at the time point.

FIG. 6. Depicts the results when Mel-2 cells were injectedsubcutaneously in the left flank (about 1 million cells/animal). Animalsreceived P28 at the indicated dose at the time of injection. FIG. 6Ashows the incidence of tumor occurrence after initiation of treatmentwith a graph indicating % of tumor free animals at days post treatmentwith Mel-2 cells. FIG. 6B shows the tumor size after initiation oftreatment with a graph indicating the average volume of the tumors (cm³)at days post treatment with Mel-2 cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “cell” includes either the singular or theplural of the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, or recovered from a pathological condition.

A used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of the condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate.

As used herein, the term “inhibit cell growth” means the slowing orceasing of cell division and/or cell expansion. This term also includesthe inhibition of cell development or increases in cell death.

As used herein, the term “inhibit angiogenesis” refers to the slowing,ceasing or reverse of the formation of blood vessels in a particularcells, tissues, or location of the body. The inhibition of angiogenesismay be due to direct or indirect effects on endothelial cells. Theinhibition may also be at any stage of the angiogenesis process. Forexample, the inhibition may be due to preventing a tumor from producingVascular Endothelial Growth Factor (VEGF), direct inhibition ofendothelial cell proliferation and/or migration, acting as an antagonistof angiogenesis growth factors, inhibition of endothelial-specificintegrin/survival signaling, or chelation of copper. The inhibition ofangiogenesis may be by any means by which the formation of blood vesselsis slowed, ceased or reversed, including any means currently used by anyanti-angiogenesis drug under development or on the market.

As used herein, the term “inappropriate angiogenesis” refers to anyoccurrence of angiogenesis that is undesirable. Inappropriateangiogenesis may be angiogenesis that is associated with a condition ina mammal. The inappropriate angiogenesis may be either the cause or thesymptom of such a condition. Inappropriate angiogenesis in a broadersense may be any angiogenesis that is unwanted, even though it may bewithin the realm of normal mammalian physiology.

A “therapeutically effective amount” is an amount effective to prevent,lower, stop or reverse the development of, or to partially or totallyalleviate the existing symptoms of a particular condition for which thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art.

The term “substantially pure,” as used herein, when used to modify aprotein or other cellular product of the invention, refers to, forexample, a protein isolated from the growth medium or cellular contents,in a form substantially free of, or unadulterated by, other proteinsand/or active inhibitory compounds. The term “substantially pure” refersto a factor in an amount of at least about 75%, by dry weight, ofisolated fraction, or at least “75% substantially pure.” Morespecifically, the term “substantially pure” refers to a compound of atleast about 85%, by dry weight, active compound, or at least “85%substantially pure.” Most specifically, the term “substantially pure”refers to a compound of at least about 95%, by dry weight, activecompound, or at least “95% substantially pure.” The term “substantiallypure” may also be used to modify a synthetically made protein orcompound of the invention, where, for example, the synthetic protein isisolated from the reagents and by-products of the synthesis reaction(s).

The term “pharmaceutical grade,” as used herein, when referring to apeptide or compound of the invention, is a peptide or compound that isisolated substantially or essentially from components which normallyaccompany the material as it is found in its natural state, includingsynthesis reagents and by-products, and substantially or essentiallyisolated from components that would impair its use as a pharmaceutical.For example, a “pharmaceutical grade” peptide may be isolated from anycarcinogen. In some instances, “pharmaceutical grade” may be modified bythe intended method of administration, such as “intravenouspharmaceutical grade,” in order to specify a peptide or compound that issubstantially or essentially isolated from any substance that wouldrender the composition unsuitable for intravenous administration to apatient. For example, an “intravenous pharmaceutical grade” peptide maybe isolated from detergents, such as SDS, and anti-bacterial agents,such as azide.

The terms “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated peptides in accordance with the inventionpreferably do not contain materials normally associated with thepeptides in their in situ environment. An “isolated” region refers to aregion that does not include the whole sequence of the polypeptide fromwhich the region was derived. An “isolated” nucleic acid, protein, orrespective fragment thereof has been substantially removed from its invivo environment so that it may be manipulated by the skilled artisan,such as but not limited to nucleotide sequencing, restriction digestion,site-directed mutagenesis, and subcloning into expression vectors for anucleic acid fragment as well as obtaining the protein or proteinfragment in substantially pure quantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. Thus, a variant peptide maybe made by manipulation of genes encoding the polypeptide. A variant maybe made by altering the basic composition or characteristics of thepolypeptide, but not at least some of its fundamental activities. Forexample, a “variant” of azurin can be a mutated azurin that retains itsability to inhibit the growth of mammalian cancer cells. In some cases,a variant peptide is synthesized with non-natural amino acids, such asε-(3,5-dinitrobenzoyl)-Lys residues. Ghadiri & Fernholz, J. Am. Chem.Soc., 112:9633-9635 (1990). In some embodiments, the variant has notmore than 20 amino acids replaced, deleted or inserted compared towild-type peptide. In some embodiments, the variant has not more than 15amino acids replaced, deleted or inserted compared to wild-type peptide.In some embodiments, the variant has not more than 10 amino acidsreplaced, deleted or inserted compared to wild-type peptide. In someembodiments, the variant has not more than 6 amino acids replaced,deleted or inserted compared to wild-type peptide. In some embodiments,the variant has not more than 5 amino acids replaced, deleted orinserted compared to wild-type peptide. In some embodiments, the varianthas not more than 3 amino acids replaced, deleted or inserted comparedto wild-type peptide.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, twothree or more carbon atoms, typically one (α) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can, for example, be a chemically modified azurinthat retains its ability to inhibit angiogenesis in mammalian cells.Chemical modifications of interest include, but are not limited to,amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation or glycosylation of the peptide. Inaddition, a derivative peptide maybe a fusion of a polypeptide orfragment thereof to a chemical compound, such as but not limited to,another peptide, drug molecule or other therapeutic or pharmaceuticalagent or a detectable probe.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a polypeptide that are identicalwith amino acid residues in a candidate sequence when the two sequencesare aligned. To determine % amino acid identity, sequences are alignedand if necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. In aspecific embodiment, Blastp (available from the National Center forBiotechnology Information, Bethesda Md.) is used using the defaultparameters of long complexity filter, expect 10, word size 3, existence11 and extension 1.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=X/Y*100

where

X is the number of amino acid residues scored as identical matches bythe sequence alignment program's or algorithm's alignment of A and B and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing truncated peptides to thecorresponding wild-type polypeptide, the truncated peptide will be the“B” sequence.

General

The present invention provides compositions comprising cupredoxin, andvariants, derivatives and structural equivalents of cupredoxins, andmethods to inhibit angiogenesis and/or inhibit the growth of cancercells in mammals. The present invention specifically relates tocompositions comprising cupredoxin, and their use in inhibiting theinappropriate angiogenesis that is associated with cancer and otherconditions. The invention also relates to variants, derivatives andstructural equivalents of cupredoxin that retain the ability to inhibitangiogenesis in mammals, and in particular the angiogenesis associatedwith tumor development, and compositions comprising the same. Mostparticularly, the invention provides compositions comprising Pseudomonasaeruginosa azurin, variants, derivatives and structural equivalents ofazurin, and their use to treat patients with conditions related toinappropriate angiogenesis, and the angiogenesis related to tumordevelopment, or prevent infection in those at risk thereof. Finally, theinvention provides methods to study angiogenesis mammalian cells,tissues and animals by contacting the cells with cupredoxin, or variant,derivative or structural equivalent thereof, before or after inducingangiogenesis and determining variations in blood vessel development.

Previously, it was know that a redox protein elaborated by Pseudomonasaeruginosa, the cupredoxin azurin, selectively enters J774 cells but notnormal cells. Zaborina et al., Microbiology 146: 2521-2530 (2000).Azurin can also selectively enter human melanoma UISO-Mel-2 or humanbreast cancer MCF-7 cells. Yamada et al., PNAS 99:14098-14103 (2002);Punj et al., Oncogene 23:2367-2378 (2004). Azurin from P. aeruginosapreferentially enters J774 murine reticulum cell sarcoma cells, forms acomplex with and stabilizes the tumor suppressor protein p53, enhancesthe intracellular concentration of p53, and induces apoptosis. Yamada etal., Infection and Immunity 70:7054-7062 (2002). Detailed studies ofvarious domains of the azurin molecule showed that amino acids 50-77(P28) (SEQ ID NO: 2) represented a protein transduction domain (PTD)critical for internalization and subsequent apoptotic activity. Yamadaet al., Cell. Microbial. 7:1418-31, (2005).

Surprisingly, it is now known that synthesized P28 not only enters intoa variety of malignant cell lines (melanoma (Mel-2), MCF-7, pancreatic,astrocytoma, glioblastoma, among others), but also non-cancerous humanumbilical vein endothelial cells (HUVEC). See Example 1. P28 enters intothese cells in a temperature dependent manner, but does not enter normalcells (fibroblast, normal mammary epithelium). As HUVEC cells are knownto instigate angiogenesis in human embryos, the entry of P28 into HUVECcells prompted an examination of the effect of P28 on angiogenesis.HUVEC cells (20,000 cells) were plated on Matrigel® coated wells andincubated in media containing 0-75 μM of P28. Cultures were examinedunder light microscopy at 4 h and 24 h post-treatment. The P28 peptideinhibited capillary tube formation of the HUVEC in a dose dependentmanner, suggesting that P28 inhibits the capillary tube formation stepof angiogenesis. See Example 2. Further, P28 inhibited the migration ofHUVEC cells on Matrigel® in a scratch wound migration assay, indicatingthat P28 also inhibits the migration step of angiogenesis. See Example3. Thus, in in vitro studies with an established angiogenesis modelsystem, HUVEC cells on Matrigel®, P28 inhibits two critical steps inangiogenesis, capillary tube formation and cell migration.

Further, the cell morphology of HUVECs was also surprisingly changed bythe addition of P28 to the growth medium. The addition of P28 to HUVECsgrowing on Matrigel® prevented the normal angiogenesis-related changesin cytoskeleton and other proteins that are associated with cellmigration. See Example 4. In the paxillin detected cell, the paxillinwas mainly localized on cell surface of the control cells, however itwas more often found on F-actin fibers in the P28 treated cells (FIG.4B). In the Fak detected cells, Fak was mainly on localized cell surfaceof the control cells, while it was more often found on F-actin fibers ofthe P28 treated cells, thus creating a less flexible and less mobilecell. The cell-cell attachment proteins CD-31/PECAM-1 wereover-expressed and localized distinctly to cell-cell junctions when theHUVECs when treated with P28, thus encouraging cell-to-cell contact.Actin nucleation and branching promoting factor WASP (Wiskott AldrichSyndrome Protein) while normally found on the cell surface in HUVECsundergoing angiogenesis, localized to the nucleus in P28 treated cellsthus altering actin branching and nucleation. Finally, P28 inhibitedβ-catenin localization to the nucleus thus further inhibitingproliferation and cell migration in HIVECs. Therefore, several of themorphological hallmarks of angiogenesis in HUVECs are reduced oreliminated by the presence of P28, further indicating that P28 has adirect effect on cells undergoing angiogenesis.

P28 can specifically inhibit the growth the Mel-2 melanoma cells invitro in a concentration dependant manner. See FIG. 5. Therefore, P28 iscapable of not only entering cancer cells in a specific manner; it alsois capable of directly inhibiting their growth. Tumor developmentproceeds in association with angiogenesis. P28 inhibited the growth ofMel-2 cells transplanted subcutaneously into athymic mice in a dosedependent manner. See Example 6. The incidence of measurable (>2 mmdiameter) tumors 30 days post-treatment of 8 and 16 mg/kg wt i.p. wassignificantly lower in treatment groups, when compared to controls.Further, tumor volume was also significantly lower in animals receiving16 mg/kg P28 as compared to control. Taken together, P28 has significantanti-tumor effects due to its selective entry into tumor cells andinhibiting their proliferation, and suppressing angiogenesis related totumor development.

Due to the high degree of structural similarity between cupredoxins, itis likely that other cupredoxins will inhibit angiogenesis in mammals aswell. Such cupredoxins may be found in, for example, bacteria or plants.It is contemplated that these other cupredoxins may be used in thecompositions and methods of the invention. Further, variants,derivatives, and structural equivalents of cupredoxins that retain theability to inhibit angiogenesis in mammalian cells may also be used inthe compositions and methods of the invention. These variants andderivatives may include, but are not limited to, truncations of acupredoxin, conservative substitutions of amino acids and proteinsmodifications such as PEGylation and all-hydrocarbon stabling ofα-helices.

It also follows that other conditions related to inappropriateangiogenesis can be treated with cupredoxins, and azurin in particular.For example, Avastin® (bevacizumab, Genentech, South San Francisco,Calif.), a recombinant humanized monoclonal IgG1 antibody that binds toand inhibits the biologic activity of human vascular endothelial growthfactor (VEGF), is not only effective in reducing angiogenesis associatedwith metastatic colorectal cancer, and is also highly effective intreating the inappropriate angiogenesis associated with neovascularage-related macular degeneration. Bashshur et al., Am J Ophthalmol.142:1-9 (2006). Therefore it is likely that P28, and other cupredoxins,and variants, derivatives and structural equivalents of cupredoxins,will also inhibit inappropriate angiogenesis in conditions other thancancer, such as those associated with neovascular age-related maculardegeneration. Further, it is likely that cupredoxins, and variants,derivatives and structural equivalents of cupredoxins will be effectivein treating other conditions related in inappropriate angiogenesis, suchas, but not limited to, diabetic retinopathy, psoriasis and rheumatoidarthritis.

Compositions of the Invention

The invention provides for peptides that are variants, derivatives orstructural equivalents of cupredoxin that inhibit angiogenesis inmammalian cells, tissues and animals. The invention further provides forpeptides that are variants, derivatives or structural equivalents ofcupredoxin that inhibit the growth of mammalian cancer cells. Theinvention further provides for peptides that are variants, derivativesor structural equivalents of cupredoxin that specifically entermammalian cancer cells. In some embodiments, the peptide is isolated. Insome embodiments, the peptide is substantially pure or pharmaceuticalgrade. In other embodiments, the peptide is in a composition thatcomprises, or consists essentially of, the peptide. In another specificembodiment, the peptide does not raise an immune response in a mammal,and more specifically a human. In some embodiments, the peptide is lessthat a full length cupredoxin, and retains some of the functionalcharacteristics of the cupredoxins. Specifically, in some embodiments,the peptide retains the ability to inhibit angiogenesis in HUVECs onMatrigel®.

The invention also provides compositions comprising at least one peptidethat is a cupredoxin, or variant, derivative or structural equivalent ofa cupredoxin, specifically in a pharmaceutical composition. In specificembodiments, the pharmaceutical composition is designed of a particularmode of administration, for example, but not limited to, oral,intraperitoneal, intravenous, or intraocular. Such compositions may behydrated in water, or may be dried (such as by lyophilization) for laterhydration. Such compositions may be in solvents other than water, suchas but not limited to, alcohol.

Because of the high structural homology between the cupredoxins, it iscontemplated that cupredoxins will have the same anti-angiogenesisactivity as P28. In some embodiments, the cupredoxin is, but is notlimited to, azurin, pseudoazurin, plastocyanin, rusticyanin, auracyaninor Laz. In particularly specific embodiments, the azurin is derived fromPseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidansssp. denitrificans I, Bordetella bronchiseptica, Methylomonas sp.,Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens,Pseudomonas chlororaphis, Xylella fastidiosa or Vibrio parahaemolyticus.In a very specific embodiment, the azurin is from Pseudomonasaeruginosa. In other specific embodiments, the cupredoxin comprises anamino acid sequence that is SEQ ID NO: 1, 3-19.

The invention provides peptides that are amino acid sequence variantswhich have amino acids replaced, deleted, or inserted as compared to thewild-type cupredoxin. Variants of the invention may be truncations ofthe wild-type cupredoxin. In some embodiments, the peptide of theinvention comprises a region of a cupredoxin that is less that the fulllength wild-type polypeptide. In some embodiments, the peptide of theinvention comprises more than about 10 residues, more than about 15residues or more than about 20 residues of a truncated cupredoxin. Insome embodiments, the peptide comprises not more than about 100residues, not more than about 50 residues, not more than about 40residues, not more than about 30 residues or not more than about 20residues of a truncated cupredoxin. In some embodiments, a cupredoxinhas amino acid sequence identity to the peptide, and more specificallySEQ ID NOS: 1, 3-19, at least about 70% amino acid sequence identity, atleast about 80% amino acid sequence identity, at least about 90% aminoacid sequence identity, at least about 95% amino acid sequence identityor at least about 99% amino acid sequence identity.

In specific embodiments, the variant of cupredoxin comprises P.aeruginosa azurin residues 50-77, azurin residues 50-67, or azurinresidues 36-88. In other embodiments, the variant of cupredoxin consistsof P. aeruginosa azurin residues 50-77, azurin residues 50-67, or azurinresidues 36-88. In other specific embodiments, the variant consists ofthe equivalent residues of a cupredoxin other that azurin. It is alsocontemplated that other cupredoxin variants can be designed that have asimilar activity to azurin residues 50-77, azurin residues 50-67, orazurin residues 36-88. To do this, the subject cupredoxin amino acidsequence will be aligned to the Pseudomonas aeruginosa azurin sequenceusing BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residueslocated on the P. aeruginosa azurin amino acid sequence, and theequivalent residues found on the subject cupredoxin sequence, and theequivalent peptide thus designed.

In one embodiment of the invention, the cupredoxin variant contains atleast amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus(SEQ ID NO: 20). In another embodiment of the invention, the cupredoxinvariant contains at least amino acids 51-77 of Pseudomonas syringaeazurin (SEQ ID NO: 21). In another embodiment of the invention, thecupredoxin variant contains at least amino acids 89-115 of Neisseriameningitidis Laz (SEQ ID NO: 22). In another embodiment of theinvention, the cupredoxin variant contains at least amino acids 52-78 ofVibrio parahaemolyticus azurin (SEQ ID NO: 23). In another embodiment ofthe invention, the cupredoxin variant contains at least amino acids51-77 of Bordetella bronchiseptica azurin (SEQ ID NO: 24).

The variants also include peptides made with synthetic amino acids notnaturally occurring. For example, non-naturally occurring amino acidsmay be integrated into the variant peptide to extend or optimize thehalf-life of the composition in the bloodstream. Such variants include,but are not limited to, D,L-peptides (diastereomer), (for example Futakiet al., J. Biol. Chem. 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al, Biochem. Pharmacol. 36(1):169-76,(1987).; peptides containing unusual amino acids (for example Lee etal., J. Pept. Res. 63(2):69-84 (2004)), olefin-containing non-naturalamino acid followed by hydrocarbon stapling (for example Schafmeister etal., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science305:1466-1470 (2004)), and peptides comprisingε-(3,5-dinitrobenzoyl)-Lys residues.

In other embodiments, the peptide of the invention is a derivative of acupredoxin. The derivatives of cupredoxin are chemical modifications ofthe peptide such that the peptide still retains some of its fundamentalactivities. For example, a “derivative” of azurin can be a chemicallymodified azurin that retains its ability to inhibit angiogenesis inmammalian cells, tissues or animals. Chemical modifications of interestinclude, but are not limited to, hydrocarbon stabling, amidation,acetylation, sulfation, polyethylene glycol (PEG) modification,phosphorylation and glycosylation of the peptide. In addition, aderivative peptide maybe a fusion of a cupredoxin, or variant,derivative or structural equivalent thereof to a chemical compound, suchas but not limited to, another peptide, drug molecule or othertherapeutic or pharmaceutical agent or a detectable probe. Derivativesof interest include chemical modifications by which the half-life in thebloodstream of the peptides and compositions of the invention can beextended or optimized, such as by several methods well known to those inthe art, including but not limited to, circularized peptides (forexample Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et al., J.Pept. Res. 63(5):409-19 (2004)), N- and C-terminal modifications (forexample Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), andolefin-containing non-natural amino acid followed by hydrocarbonstapling (for example Schafmeister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)).

In another embodiment, the peptide is a structural equivalent of acupredoxin. Examples of studies that determine significant structuralhomology between cupredoxins and other proteins include Toth et al.(Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a cupredoxin and the structural equivalentis determined by using the VAST algorithm. Gibrat et al., Curr OpinStruct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995).In specific embodiments, the VAST p value from a structural comparisonof a cupredoxin to the structural equivalent is less than about 10⁻³,less than about 10⁻⁵, or less than about 10⁻⁷. In other embodiments,significant structural homology between a cupredoxin and the structuralequivalent is determined by using the DALI algorithm. Holm & Sander, J.Mol. Biol. 233:123-138 (1993). In specific embodiments, the DALI Z scorefor a pairwise structural comparison is at least about 3.5, at leastabout 7.0, or at least about 10.0.

It is contemplated that the peptides of the composition of invention maybe more than one of a variant, derivative and/or structural equivalentof a cupredoxin. For example, the peptides may be a truncation of azurinthat has been PEGylated, thus making it both a variant and a derivative.In one embodiment, the peptides of the invention are synthesized withα,α-disubstituted non-natural amino acids containing olefin-bearingtethers, followed by an all-hydrocarbon “staple” by ruthenium catalyzedolefin metathesis. Scharmeister et al., J. Am. Chem. Soc. 122:5891-5892(2000); Walensky et al., Science 305:1466-1470 (2004). Additionally,peptides that are structural equivalents of azurin may be fused to otherpeptides, thus making a peptide that is both a structural equivalent anda derivative. These examples are merely to illustrate and not to limitthe invention. Variants, derivatives or structural equivalents ofcupredoxin may or may not bind copper.

In some embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof has some of the functional characteristicsof the P. aeruginosa azurin, and specifically P28. In a specificembodiment, the cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that may inhibit angiogenesis in mammaliancells, tissues or animals, and specifically but not limited to, HUVECs.The invention also provides for the cupredoxins and variants,derivatives and structural equivalents of cupredoxin that may have theability to inhibit the growth of mammalian cancer cells, andspecifically but not limited to, melanoma, breast, pancreas,glioblastoma, astrocytoma, or lung cancer cells. The invention alsoprovides for the cupredoxins and variants, derivatives and structuralequivalents of cupredoxin that may have the ability to enter mammaliancancer cells as compared to equivalent non-cancer cells, specifically,but not limited to, melanoma, breast, pancreas, glioblastoma,astrocytoma, or lung cancer cells. Inhibition of angiogenesis or growthof cancer cells is any decrease, or lessening of the rate of increase,of that activity that is statistically significant as compared tocontrol treatments. The entry into cancer cells may be the entry intothe cancer cells that is statistically significant when compared to therate of entry into equivalent normal cells.

Because it is now known that cupredoxins can inhibit angiogenesis inmammalian cells, tissues or animals, and specifically HUVECs growing onMatrigel®, it is now possible to design variants and derivatives ofcupredoxins that retain this anti-angiogenesis activity. Such variants,derivatives and structural equivalents can be made by, for example,creating a “library” of various variants, derivatives and structuralequivalents of cupredoxins and then testing each for anti-angiogenesisactivity, and specifically anti-angiogenesis in HUVECs using one of manymethods known in the art, such the exemplary method in Examples 2 and 3.It is contemplated that the resulting variants and derivatives ofcupredoxins with anti-angiogenesis activity can be used in the methodsof the invention, in place of or in addition to cupredoxins.

In some specific embodiments, the cupredoxin or variant, derivative orstructural equivalent inhibits capillary tube formation in HUVEC cellsto a degree that is statistically different from a non-treated control.A peptide can be tested for this activity by using the capillary tubeformation test described in Example 3 or in Sulochana et al., J. Biol.Chem. 280:27936-27948 (2005). Other methods to determine whethercapillary tube formation is inhibited another are well known in the artand may be used as well.

In some specific embodiments, the cupredoxin or variant, derivative orstructural equivalent inhibits HUVEC migration in a scratch woundmigration assay to a degree that is statistically different from anon-treated control. A peptide can be tested for this activity by usingthe capillary tube formation test described in Example 4. Other methodsto determine whether HUVEC migration is inhibited are well known in theart and may be used as well.

Cupredoxins

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial electron transferchains or are of unknown function. The copper ion is solely bound by theprotein matrix. A special distorted trigonal planar arrangement to twohistidine and one cysteine ligands around the copper gives rise to verypeculiar electronic properties of the metal site and an intense bluecolor. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution.

The cupredoxins in general have a low sequence homology but highstructural homology. Gough & Clothia, Structure 12:917-925 (2004); DeRienzo et al., Protein Science 9:1439-1454 (2000). For example, theamino acid sequence of azurin is 31% identical to that of auracyanin B,16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3%to that of pseudoazurin. See, Table 1. However, the structuralsimilarity of these proteins is more pronounced. The VAST p value forthe comparison of the structure of azurin to auracyanin B is 10^(−7.4),azurin to rusticyanin is 10⁻⁵, azurin to plastocyanin is 10^(−5.6), andazurin to psuedoazurin is 10^(−4.1).

All of the cupredoxins possess an eight-stranded Greek key beta-barrelor beta-sandwich fold and have a highly conserved site architecture. DeRienzo et al., Protein Science 9:1439-1454 (2000). A prominenthydrophobic patch, due to the presence of many long chain aliphaticresidues such as methionines and leucines, is present around the coppersite in azurins, amicyanins, cyanobacterial plastocyanins, cucumberbasic protein and to a lesser extent, pseudoazurin and eukaryoticplastocyanins. Id. Hydrophobic patches are also found to a lesser extentin stellacyanin and rusticyanin copper sites, but have differentfeatures. Id.

TABLE 1 Sequence and structure alignment of azurin (1JZG) from P.aeruginosa to other proteins using VAST algorithm. Alignment % aa P- PDBlength¹ identity value² Score^(3i)) RMSD⁴) Description 1AOZ A 2 82 18.310e−7 12.2 1.9 Ascorbate oxidase 1QHQ_A 113 31 10e−7.4 12.1 1) 1.9 2)AuracyaninB 1V54 B 1 79 20.3 10e−6.0 11.2 2.1 Cytocrome c oxidase 1GY2 A92 16.3 10e−5.0 11.1 3) 1.8 4) Rusticyanin 3MSP A 74 8.1 10e−6.7 10.92.5 Motile Major Sperm Protein⁵ 1IUZ 74 20.3 10e−5.6 10.3 5) 2.3 6)Plastocyanin 1KGY E 90 5.6 10e−4.6 10.1 7) 3.4 8) Ephrinb2 1PMY 75 17.310e−4.1 9.8 9) 2.3 10)  Pseudoazurin ¹Aligned Length: The number ofequivalent pairs of C-alpha atoms superimposed between the twostructures, i.e. how many residues have been used to calculate the 3Dsuperposition. ²P-VAL: The VAST p value is a measure of the significanceof the comparison, expressed as a probability. For example, if the pvalue is 0.001, then the odds are 1000 to 1 against seeing a match ofthis quality by pure chance. The p value from VAST is adjusted for theeffects of multiple comparisons using the assumption that there are 500independent and unrelated types of domains in the MMDB database. The pvalue shown thus corresponds to the p value for the pairwise comparisonof each domain pair, divided by 500. ³Score: The VASTstructure-similarity score. This number is related to the number ofsecondary structure elements superimposed and the quality of thatsuperposition. Higher VAST scores correlate with higher similarity.⁴RMSD: The root mean square superposition residual in Angstroms. Thisnumber is calculated after optimal superposition of two structures, asthe square root of the mean square distances between equivalent C-alphaatoms. Note that the RMSD value scales with the extent of the structuralalignments and that this size must be taken into consideration whenusing RMSD as a descriptor of overall structural similarity. ⁵ C.elegans major sperm protein proved to be an ephrin antagonist in oocytematuration. Kuwabara, Genes and Development 17: 155-161 (2003).

Azurin

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin certain bacteria. The azurins include those from P. aeruginosa (PA)(SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans. Murphy et al., J.Mol. Biol. 315:859-871 (2002). The amino acid sequence identity betweenthe azurins varies between 60-90%, these proteins showed a strongstructural homology. All azurins have a characteristic β-sandwich withGreek key motif and the single copper atom is always placed at the sameregion of the protein. In addition, azurins possess an essentiallyneutral hydrophobic patch surrounding the copper site. Id.

Plastocyanins

The plastocyanins are soluble proteins of cyanobacteria, algae andplants that contain one molecule of copper per molecule and are blue intheir oxidized form. They occur in the chloroplast, where they functionas electron carriers. Since the determination of the structure of poplarplastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha,Chlamydomonas) and plant (French bean) plastocyanins has been determinedeither by crystallographic or NMR methods, and the poplar structure hasbeen refined to 1.33 Å resolution. SEQ ID NO: 3 shows the amino acidsequence of plastocyanin from Phormidium laminosum, a thermophiliccyanobacterium.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths involving plastocyanin. One putative electron transfer path isrelatively short (approximately 4 Å) and involves the solvent-exposedcopper ligand His-87 in the hydrophobic patch, while the other is morelengthy (approximately 12-15 Å) and involves the nearly conservedresidue Tyr-83 in the negative patch. Redinbo et al., J. Bioenerg.Biomembr. 26:49-66 (1994).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a Thiobacillus (now called Acidithiobacillus). The X-raycrystal structure of the oxidized form of the extremely stable andhighly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 4) has been determined by multiwavelength anomalousdiffraction and refined to 1.9 Å resolution. The rusticyanins arecomposed of a core beta-sandwich fold composed of a six- and aseven-stranded b-sheet. Like other cupredoxins, the copper ion iscoordinated by a cluster of four conserved residues (His 85, Cys138,His143, Met148) arranged in a distorted tetrahedron. Walter, R. L. etal., J. Mol. Biol. 263:730-51 (1996).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 5. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Phytocyanins

The proteins identifiable as phytocyanins include, but are not limitedto, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, acucumber peeling cupredoxin, a putative blue copper protein in pea pods,and a blue copper protein from Arabidopsis thaliana. In all exceptcucumber basic protein and the pea-pod protein, the axial methionineligand normally found at blue copper sites is replaced by glutamine.

Auracyanin

Three small blue copper proteins designated auracyanin A, auracyaninB-1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms are glycoproteins and have almost identical properties to eachother, but are distinct from the A form. The sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrates apparent monomermolecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. Van Dreissche et al.,Protein Science 8:947-957 (1999). His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 15 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. Bond et al., J. Mol. Biol. 306:47-67 (2001). With the exceptionof an additional N-terminal strand, the molecule is very similar to thatof the bacterial cupredoxin, azurin. As in other cupredoxins, one of theCu ligands lies on strand 4 of the polypeptide, and the other three liealong a large loop between strands 7 and 8. The Cu site geometry isdiscussed with reference to the amino acid spacing between the latterthree ligands. The crystallographically characterized Cu-binding domainof auracyanin B is probably tethered to the periplasmic side of thecytoplasmic membrane by an N-terminal tail that exhibits significantsequence identity with known tethers in several othermembrane-associated electron-transfer proteins. The amino acid sequencesof the B forms are presented in McManus et al. J. Biol. Chem.267:6531-6540 (1992). See exemplary amino acid sequence SEQ ID NO: 16for chain B of auracyanin from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Stellacyanin

Stellacyanins are a subclass of phytocyanins, a ubiquitous family ofplant cupredoxins. An exemplary sequence of a stellacyanin is includedherein as SEQ ID NO: 14. The crystal structure of umecyanin, astellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc.127:158-166 (2005)) and cucumber stellacyanin (Hart el al., ProteinScience 5:2175-2183 (1996)) is also known. The protein has an overallfold similar to the other phytocyanins. The ephrin B2 protein ectodomaintertiary structure bears a significant similarity to stellacyanin. Tothet al., Developmental Cell 1:83-92 (2001). An exemplary amino acidsequence of a stellacyanin is found in the National Center forBiotechnology Information Protein Data Bank as Accession No. 1JER, SEQID NO: 14.

Cucumber Basic Protein

An exemplary amino acid sequence from a cucumber basic protein isincluded herein as SEQ ID NO: 17. The crystal structure of the cucumberbasic protein (CBP), a type 1 blue copper protein, has been refined at1.8 Å resolution. The molecule resembles other blue copper proteins inhaving a Greek key beta-barrel structure, except that the barrel is openon one side and is better described as a “beta-sandwich” or “beta-taco”.Guss et al., J. Mol. Biol. 262:686-705 (1996). The ephrinB2 proteinectodomian tertiary structure bears a high similarity (rms deviation 1.5Å for the 50α carbons) to the cucumber basic protein. Toth et al.,Developmental Cell 1:83-92 (2001).

The Cu atom has the normal blue copper NNSS′ co-ordination with bondlengths Cu—N(His39)=1.93 A, Cu—S(Cys79)=2.16 A, Cu—N(His84)=1.95 A,Cu—S(Met89)=2.61 A. A disulphide link, (Cys52)-S—S-(Cys85), appears toplay an important role in stabilizing the molecular structure. Thepolypeptide fold is typical of a sub-family of blue copper proteins(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3,with which CBP has a high degree of sequence identity. The proteinscurrently identifiable as phytocyanins are CBP, stellacyanin,mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative bluecopper protein in pea pods, and a blue copper protein from Arabidopsisthaliana. In all except CBP and the pea-pod protein, the axialmethionine ligand normally found at blue copper sites is replaced byglutamine. An exemplary sequence for cucumber basic protein is found inNCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.

Methods of Use

The invention provides methods to treat a mammalian patient sufferingfrom cancer, recovering from cancer, recovered from cancer or at risk toget cancer comprising administering to the patient at least onepolypeptide that is a cupredoxin, or variant, derivative or structuralequivalent thereof, as described above. Specifically, cancers that maybe treated with the compositions of the invention include, but are notlimited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, orlung cancer. The invention further provides methods to treat patientssuffering from, recovering from, recovered from or at risk of gettingother conditions related to inappropriate angiogenesis comprisingadministering to the patient at least one polypeptide that is acupredoxin, or variant, derivative or structural equivalent thereof.These conditions include, but are not limited to, neovascularage-related macular degeneration, diabetic retinopathy, psoriasis andrheumatoid arthritis. In specific embodiments, the patient is human.

The invention further includes methods to study angiogenesis comprisingcontacting mammalian cells with a composition comprising cupredoxin, orvariant, derivative or structural equivalent thereof. In someembodiments, the cells are HUVECs, while in others they are other cellsthat undergo angiogenesis. The methods of the invention further includemethods to study conditions related to inappropriate angiogenesiscomprising contacting mammalian cells with a composition comprisingcupredoxin, or variant, derivative or structural equivalent thereof. Inthese methods, cells may be those which undergo angiogenesis inmammalian patients suffering from the condition.

The compositions comprising a cupredoxin or variant, derivative orstructural equivalent thereof can be administered to the patient by manyroutes and in many regimens that will be well known to those in the art.In specific embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof is administered intravenously,intramuscularly, subcutaneously or intraoccularly. The compositions maybe administered to the patient by any means that delivers the peptidesto the site of inappropriate angiogenesis.

In one embodiment, the methods may comprise co-administering to apatient one unit dose of a composition comprising a cupredoxin or avariant, derivative or structural equivalent of cupredoxin and one unitdose of a composition comprising another anti-cancer drug, in eitherorder, administered at about the same time, or within about a given timefollowing the administration of the other, for example, about one minuteto about 60 minutes following the administration of the other drug, orabout 1 hour to about 12 hours following the administration of the otherdrug. Such drugs include, for example, those listed herein andspecifically 5-fluorouracil; Interferon α; Methotrexate; Tamoxifen; andVincrinstine. The above examples are provided for illustration only,many other such compounds are known to those skilled in the art. Thecompounds of the invention may also be used in conjunction withradiation therapy and surgery.

Other drugs suitable for treating cancer include, but not limited to,alkylating agents such as nitrogen mustards, alkyl sulfonates,nitrosoureas, ethylenimines, and triazenes; antimetabolites such asfolate antagonists, purine analogues, and pyrimidine analogues;antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin,and plicamycin; enzymes such as L-asparaginase; farnesyl-proteintransferase inhibitors; 5.alpha.-reductase inhibitors; inhibitors of17.beta.-hydroxysteroid dehydrogenase type 3; hormonal agents such asglucocorticoids, estrogens/antiestrogens, androgens/antiandrogens,progestins, and luteinizing hormone-releasing hormone antagonists,octreotide acetate; microtubule-disruptor agents, such as ecteinascidinsor their analogs and derivatives; microtubule-stabilizing agents such astaxanes, for example, paclitaxel (Taxol™), docetaxel (Taxotere™), andtheir analogs, and epothilones, such as epothilones A-F and theiranalogs; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, taxanes; and topiosomerase inhibitors;prenyl-protein transferase inhibitors; and miscellaneous agents such ashydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinumcoordination complexes such as cisplatin and carboplatin; and otheragents used as anti-cancer and cytotoxic agents such as biologicalresponse modifiers, growth factors; immune modulators and monoclonalantibodies.

Representative examples of these classes of anti-cancer and cytotoxicagents include but are not limited to mechlorethamine hydrochloride,cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan,carmustin, lomustine, semustine, streptozocin, thiotepa, dacarbazine,methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin,cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride,daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D,safracins, saframycins, quinocarcins, discodermolides, vincristine,vinblastine, vinorelbine tartrate, etopo side, etoposide phosphate,teniposide, paclitaxel, tamoxifen, estramustine, estramustine phosphatesodium, flutamide, buserelin, leuprolide, pteridines, diyneses,levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim,sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride,betamethosone, gemcitabine hydrochloride, altretamine, and topoteca andany analogs or derivatives thereof.

Preferred members of these classes include, but are not limited to,paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin,daunorubicin, aminopterin, methotrexate, methopterin, mitomycin C,ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine,gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxinderivatives such as etoposide, etoposide phosphate or teniposide,melphalan, vinblastine, vincristine, leurosidine, vindesine andleurosine.

Examples of anticancer and other cytotoxic agents useful toco-administer with the compositions of the invention include thefollowing: epothilone derivatives as found in German Patent No.4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclindependent kinase inhibitors as found in WO 99/24416 (see also U.S. Pat.No. 6,040,321); and prenyl-protein transferase inhibitors as found in WO97/30992 and WO 98/54966; and agents such as those described genericallyand specifically in U.S. Pat. No. 6,011,029 (the compounds of which canbe employed together with any NHR modulators (including, but not limitedto, those of present invention) such as AR modulators, ER modulators,with LHRH modulators, or with surgical techniques.

In another embodiment, the cupredoxin, variant, derivative or structuralequivalent thereof may be co-administered with a drug for the treatmentof neovascular age-related macular degeneration. Such drugs include, butare not limited to, Lucentis® (Genetech, South San Francisco Calif.,Ranibizumab, vitreous injection), Macugen® (OSI Pharmaceuticals,Melville N.Y., pegaptanib, vitreous injection), Retaane® (Alcon, FortWorth Tex., Anecortave, posterior juxtascleral injection), AdPEDF(GenVec, Gaithersburg Md., anti-angiogenic gene therapy, intravitreal orsub-Tenon injection), EVIZON® (Genaera, Plymouth Meeting, Pa.,anti-angiogenic aminosterol, Squalamine, intravenous injection),Combretastatin AAdPEDF4 Prodrug (OXiGENE, Waltham Mass., CA4P, VascularTargeting agentc), Cand5 (Acuity Pharmaceuticals, Inc., PhiladelphiaPa., siRNA targeting Vascular Endothelial Growth Factor (VEGF),Sirna-027® (Sirna Therapeutics, San Francisco, Calif., siRNA targetingVascular Endothelial Growth Factor Receptor-1 (VEGFR-1)), Celecoxib withPDT (Celebrex®, Oral anti-inflammatory drug), and Envision® TD (ControlDelivery Systems, Watertown, Mass., fluocinolone implant sustainedrelease steroid implant in vitreous).

In another embodiment, the cupredoxin, variant, derivative or structuralequivalent thereof may be co-administered with a drug for the treatmentof diabetic retinopathy. Surgical treatment is also contemplated as aco-treatment with the compositions of the invention.

In another embodiment, the cupredoxin, variant, derivative or structuralequivalent thereof may be co-administered with a drug for the treatmentof psoriasis. Such drugs include, but are not limited to, Amevive®,Raptiva®, Enbrel®, Humira®, Remicade®, Cyclosporine, Neoral®,Methotrexate, Soriatane®, Accutane®, Hydrea®, mycophenolate mofetil,sulfasalazine, and 6-Thioguanine.

In another embodiment, the cupredoxin, variant, derivative or structuralequivalent thereof may be co-administered with a drug for the treatmentof rheumatoid arthritis. Such drugs include, but are not limited to,Methotrexate (Rheumatrex®, Folex PFS®), Sulfasalazine (Azulfidine®),Leflunomide (Arava®), Gold salts (aurothiomalate, auranofin [Ridaura®]),D-penicillamine, Hydroxychloroquine (Plaquenil®), Azathioprine(Imuran®), Cyclosporine (Neoral®), Etanercept (Enbrel®), Infliximab(Remicade®), Adalimumab (Humira®), Anakinra (Kineret®), Abatacept(Orencia®), Prednisone (Deltasone®, Meticorten®, Orasone®),Betamethasone (Celestone®), Nonsteroidal anti-inflammatory drugs(NSAIDs), and COX-2 inhibitors (celecoxib, Celebrex®).

Pharmaceutical Compositions Comprising Cupredoxin, or Variant,Derivative or Structural Equivalent Thereof

Pharmaceutical compositions comprising cupredoxin or variant, derivativeor structural equivalents thereof, can be manufactured in anyconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The substantially pure or pharmaceutical gradecupredoxin or variants, derivatives and structural equivalents thereofcan be readily combined with a pharmaceutically acceptable carrierwell-known in the art. Such carriers enable the preparation to beformulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable carriers or excipients canalso include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders. In some embodiments, the pharmaceutical preparation issubstantially free of preservatives. In other embodiments, thepharmaceutical preparation may contain at least one preservative.General methodology on pharmaceutical dosage forms is found in Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems (LippencottWilliams & Wilkins, Baltimore Md. (1999)).

The composition comprising a cupredoxin or variant, derivative orstructural equivalent thereof used in the invention may be administeredin a variety of ways, including by injection (e.g., intradermal,subcutaneous, intramuscular, intraperitoneal and the like), byinhalation, by topical administration, by suppository, by using atransdermal patch or by mouth. General information on drug deliverysystems can be found in Ansel et al., Id. In some embodiments, thecomposition comprising a cupredoxin or variant, derivative or structuralequivalent thereof can be formulated and used directly as injectibles,for subcutaneous and intravenous injection, among others. The injectableformulation, in particular, can advantageously be used to treat patientsthat are at risk of, likely to have or have a condition related toinappropriate angiogenesis. The composition comprising a cupredoxin orvariant, derivative or structural equivalent thereof can also be takenorally after mixing with protective agents such as polypropylene glycolsor similar coating agents.

When administration is by injection, the cupredoxin or variant,derivative or structural equivalent thereof may be formulated in aqueoussolutions, specifically in physiologically compatible buffers such asHanks solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the cupredoxin or variant,derivative or structural equivalent thereof may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. In some embodiments, the pharmaceutical composition does notcomprise an adjuvant or any other substance added to enhance the immuneresponse stimulated by the peptide. In some embodiments, thepharmaceutical composition comprises a substance that inhibits an immuneresponse to the peptide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the cupredoxin or variant, derivative or structuralequivalent thereof may be composed of crystalloids or colloids.Crystalloids as used herein are aqueous solutions of mineral salts orother water-soluble molecules. Colloids as used herein contain largerinsoluble molecules, such as gelatin. Intravenous fluids may be sterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 2.

TABLE 2 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 2/3 & 1/3 3.3% Dextrose/51 51 168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normalsaline 0.9% NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution*Ringer's lactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/LCa²⁺.

When administration is by inhalation, the cupredoxin or variant,derivative or structural equivalent thereof may be delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the proteins and a suitable powderbase such as lactose or starch.

When administration is by topical administration, the cupredoxin orvariant, derivative or structural equivalent thereof may be formulatedas solutions, gels, ointments, creams, jellies, suspensions, and thelike, as are well known in the art. In some embodiments, administrationis by means of a transdermal patch. When administration is bysuppository (e.g., rectal or vaginal), cupredoxin or variants andderivatives thereof compositions may also be formulated in compositionscontaining conventional suppository bases.

When administration is oral, a cupredoxin or variant, derivative orstructural equivalent thereof can be readily formulated by combining thecupredoxin or variant, derivative or structural equivalent thereof withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the cupredoxin and variants,derivatives or structural equivalent thereof to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, cellulosepreparation, granulating agents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a cupredoxin or variant,derivative or structural equivalent thereof allow for the release ofcupredoxin or variant, derivative or structural equivalent thereof overextended periods of time, such that without the sustained releaseformulation, the cupredoxin or variant, derivative or structuralequivalent thereof would be cleared from a subject's system, and/ordegraded by, for example, proteases and simple hydrolysis beforeeliciting or enhancing a therapeutic effect.

The half-life in the bloodstream of the peptides of the invention can beextended or optimized by several methods well known to those in the art.The peptide variants of the invention may include, but are not limitedto, various variants that may increase their stability, specificactivity, longevity in the bloodstream, and/or decrease immunogenicityof the cupredoxin, while retaining the ability of the peptide to inhibitangiogenesis, to enter mammal cancer cells and/or inhibit the growth ofmammalian cancer cells. Such variants include, but are not limited to,those which decrease the hydrolysis of the peptide, decrease thedeamidation of the peptide, decrease the oxidation, decrease theimmunogenicity, increase the structural stability of the peptide orincrease the size of the peptide. Such peptides also includecircularized peptides (see Monk et al., BioDrugs 19(4):261-78, (2005);DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), D,L-peptides(diastereomer), Futaki et al., J. Biol. Chem. February 23;276(8):5836-40 (2001); Papo et al., Cancer Res. 64(16):5779-86 (2004);Miller et al., Biochem. Pharmacol. 36(1):169-76, (1987)); peptidescontaining unusual amino acids (see Lee et al., J. Pept. Res.63(2):69-84 (2004)), N- and C-terminal modifications (see Labrie et al.,Clin. Invest. Med. 13(5):275-8, (1990)), hydrocarbon stapling (seeSchafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); Walenski etal., Science 305:1466-1470 (2004)) and PEGylation. Of particularinterest are d-isomerization (substitution) and modification of peptidestability via D-substitution or L-amino acid substitution.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

Administration of Cupredoxin or Variant, Derivative or StructuralEquivalent Thereof

The cupredoxin or variant, derivative or structural equivalent thereofcan be administered formulated as pharmaceutical compositions andadministered by any suitable route, for example, by oral, buccal,inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary) or vitreousadministration. The pharmaceutical formulations thereof can beadministered in any amount effective to achieve its intended purpose.More specifically, the composition is administered in a therapeuticallyeffective amount. In specific embodiments, the therapeutically effectiveamount is generally from about 0.01-20 mg/day/kg of body weight.

The compounds comprising cupredoxin or variant, derivative or structuralequivalent thereof are useful for the treatment and/or prophylaxis ofconditions related in inappropriate angiogenesis, alone or incombination with other active agents. The appropriate dosage will, ofcourse, vary depending upon, for example, the compound of cupredoxin orvariant, derivative or structural equivalent thereof employed, the host,the mode of administration and the nature and severity of the conditionsbeing treated. However, in general, satisfactory results in humans areindicated to be obtained at daily dosages from about 0.01-20 mg/kg ofbody weight. An indicated daily dosage in humans is in the range fromabout 0.7 mg to about 1400 mg of a compound of cupredoxin or variant,derivative or structural equivalent thereof conveniently administered,for example, in daily doses, weekly doses, monthly doses, and/orcontinuous dosing. Daily doses can be in discrete dosages from 1 to 12times per day. Alternatively, doses can be administered every other day,every third day, every fourth day, every fifth day, every sixth day,every week, and similarly in day increments up to 31 days or over.Alternatively, dosing can be continuous using patches, i.v.administration and the like.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin or variant, derivative or structuralequivalent thereof which are sufficient to maintain therapeutic effect.Generally, the desired cupredoxin or variant, derivative or structuralequivalent thereof is administered in an admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

In one aspect, the cupredoxin or variant, derivative or structuralequivalent thereof is delivered as DNA such that the polypeptide isgenerated in situ. In one embodiment, the DNA is “naked,” as described,for example, in Ulmer et al., (Science 259:1745-1749 (1993)) andreviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of nakedDNA may be increased by coating the DNA onto a carrier, e.g.,biodegradable beads, which are then efficiently transported into thecells. In such methods, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are essential for propagation in anappropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cupredoxin. In expression vectors, the introducedDNA is operably-linked to elements such as promoters that signal to thehost cell to highly transcribe the inserted DNA. Some promoters areexceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acupredoxin and variants and derivatives thereof polynucleotide to aninducible promoter can control the expression of the cupredoxin andvariants and derivatives thereof in response to specific factors.Examples of classic inducible promoters include those that areresponsive to α-interferon, heat shock, heavy metal ions, and steroidssuch as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511 (1990))and tetracycline. Other desirable inducible promoters include those thatare not endogenous to the cells in which the construct is beingintroduced, but, are responsive in those cells when the induction agentis exogenously supplied. In general, useful expression vectors are oftenplasmids. However, other forms of expression vectors, such as viralvectors (e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, polylinker sites,enhancer elements, promoters, and transcription termination sequences.

Kits Comprising Cupredoxin, or Variant, Derivative or StructuralEquivalent Thereof

In one aspect, the invention provides regimens or kits comprising one ormore of the following in a package or container: (1) a biologicallyactive composition comprising at least one cupredoxin or variant,derivative or structural equivalent thereof; (2) an anti-viral oranti-bacterial drug, specifically an anti-cancer drug, an anti-maculardegeneration drug, an anti psoriasis drug or an anti-rheumatoidarthritis drug.

When a kit is supplied, the different components of the composition maybe packaged in separate containers, if appropriate, and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized cupredoxin andvariants, derivatives and structural equivalents thereof, or buffersthat have been packaged under a neutral, non-reacting gas, such asnitrogen. Ampules may consist of any suitable material, such as glass,organic polymers, such as polycarbonate, polystyrene, etc., ceramic,metal or any other material typically employed to hold similar reagents.Other examples of suitable containers include simple bottles that may befabricated from similar substances as ampules, and envelopes, that maycomprise foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, or thelike. Containers may have a sterile access port, such as a bottle havinga stopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to be mixed.Removable membranes may be glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

Modification of Cupredoxin and Variants, Derivatives and StructuralEquivalents Thereof

Cupredoxin or variant, derivative or structural equivalents thereof maybe chemically modified or genetically altered to produce variants andderivatives as explained above. Such variants and derivatives may besynthesized by standard techniques.

In addition to naturally-occurring allelic variants of cupredoxin,changes can be introduced by mutation into cupredoxin coding sequencethat incur alterations in the amino acid sequences of the encodedcupredoxin that do not significantly alter the ability of cupredoxin toinhibit angiogenesis. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequences of the cupredoxinwithout altering biological activity, whereas an “essential” amino acidresidue is required for such biological activity. For example, aminoacid residues that are conserved among the cupredoxins are predicted tobe particularly non-amenable to alteration, and thus “essential.”

Amino acids for which conservative substitutions that do not change theactivity of the polypeptide can be made are well known in the art.Useful conservative substitutions are shown in Table 3, “Preferredsubstitutions.” Conservative substitutions whereby an amino acid of oneclass is replaced with another amino acid of the same type fall withinthe scope of the invention so long as the substitution does notmaterially alter the biological activity of the compound.

TABLE 3 Preferred substitutions Original Preferred residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the cytotoxic factor function. Residues aredivided into groups based on common side-chain properties as denoted inTable 4. Non-conservative substitutions entail exchanging a member ofone of these classes for another class. Substitutions may be introducedinto conservative substitution sites or more specifically intonon-conserved sites.

TABLE 4 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol.154:329-350 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on the cloned DNA to produce the cupredoxinvariant DNA.

Known mutations of cupredoxins can also be used to create variantcupredoxin to be used in the methods of the invention. For example, theC112D and M44KM64E mutants of azurin are known to have cytotoxic andgrowth arresting activity that is different from the native azurin, andsuch altered activity can be useful in the treatment methods of thepresent invention. One embodiment of the methods of the inventionutilizes cupredoxin and variants and derivatives thereof retaining theability inhibit angiogenesis. In another embodiment, the methods of thepresent invention utilize cupredoxin variants such as the M44KM64Emutant, having the ability to cause cellular growth arrest.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof, and,therefore, only such limitations should be imposed as are indicated bythe appended embodiments.

EXAMPLES Example 1 Entry of P28 into Human Umbilical Vein EndothelialCells

P28 was labeled with 20 μM Alexafluor® 568 (Molecular Probes, Eugene,Oreg.). Indicated cell lines were cultured on cell culture coated coverslips overnight at 37° C. Pre-warmed media containing labeled peptidewas added at indicated concentrations. After incubation with the labeledpeptide, the cover slips were washed 3× with PBS and fixed in formalinfor 5 minutes. Cover slips were then mounted in media containing 1.5 μgml⁻¹ DAPI for nuclear staining (VECTASHIELD®, Vector Laboratories,Burlingame, Calif.). Analysis was performed with a confocal microscope(Model LC510, Carl Zeiss, Thornwood, N.Y.).

P28 effectively entered malignant cell lines originating from melanoma,breast, pancreas, glioblastoma, astrocytoma, and lung (FIG. 1A). P28 wasalso efficiently entered HUVEC cells (FIG. 1C). No significant entry wasobserved in other “normal” cell lines originating from skin fibroblasts,breast and pancreas FIG. 1B). Therefore, in addition to specificallyentering mammalian cancer cells, P28 also specifically enters HUVECcells.

This experiment shows that the P. aeruginosa azurin 50-77 peptide hasactivity that inhibits capillary tube formation in endothelial cells,one step in angiogenesis. The P. aeruginosa azurin 50-77 peptide cantherefore be used to control angiogenesis and hence be utilized as acancer treatment, and treatment of other conditions related toinappropriate angiogenesis.

Example 2 Effects of P28 on HUVEC Capillary Tube Formation on Matrigel®

Matrigel® Matrix (Becton Dickinson Biosciences, San Jose Calif.) is asolubulized basement membrane preparation extracted from EHS mousesarcoma, a tumor rich in ECM proteins. Its major component is laminin,followed by collagen IV, heparan sulfate proteoglycans, and entactin 1.At room temperature, Matrigel® Matrix polymerizes to producebiologically active matrix material resembling the mammalian cellularbasement membrane. Cells behave as they do in vivo when they arecultured on Matrigel® Matrix. It provides a physiologically relevantenvironment for studies of cell morphology, biochemical function,migration or invasion, and gene expression. Matrigel® Matrix serves as asubstrate for in vitro endothelial cell invasion and tube formationassays.

The effects of P28 on the capillary tube formation of HUVEC cells wereinvestigated using Matrigel®. HUVEC cells were plated (15,000cells/well) on Matrigel® coated 8 well chamber slides with 20 ng/ml VEGFand in the presence or absence of peptide. P28 concentrations of 0 μM(control), 0.10 μM, 0.30 μM, 0.92 μM, 2.77 μM, 8.33 μM, 25 μM and 75 μMwere used. Cells were stained 4 h and 24 h post-treatment with calceinAM, and capillary tube formation was examined using a fluorescencemicroscope (FIG. 2A). The results show that as little as 0.10 μMprevented capillary tube formation by HUVEC cells by about 50% (FIG.2A). P28 therefore inhibits tube formation of HUVEC cells, and willtherefore also inhibit the capillary tube formation related toangiogenesis.

Example 3 Effects of P28 on HUVEC Motility

The effects of P28 on HUVEC motility was investigated with the scratchwound migration assay. HUVEC cells were plated in 60 mm tissue culturedishes and allowed to reach 90% confluence. After removing the media,cell layers were wounded using a 1 ml sterile plastic pipette tip.Plates were rinsed with culture media. Media with 20 ng/ml VEGF alone ormedia with 20 ng/ml VEGF and containing P28 peptide was then added tothe plates. One dish was scratched as above and fixed immediately inorder to mark exact wound area. FIG. 3A. After 24 h, cultures were fixedand stained for F-actin and nuclei using Phalloidin and Hoechst stain.Scratched areas were examined using a florescence microscope andphotographed. The number of cells that migrated into the scratched areawas counted in the control (FIG. 3B) and peptide treated dishes (FIG.3C).

The number of HUVECs that migrated into the scratch wound in the cellstreated with P28 was about half that of those that migrated into thescratch wound in the control. Figure D. Therefore, the presence of P28inhibited the motility of HUVECs undergoing angiogenesis.

Example 4 Effects of P28 on HUVEC Structural Proteins

The effects of P28 on HUVEC structural proteins was studied to gain abetter understanding of the way P28 affects these cells. HUVEC cellsplated on Matrigel® coated cover slips were incubated with 20 ng/ml VEGFin the presence or absence of 25 μM P28 peptide for 4 h or 24 h. Afterincubation, cells were rinsed in PBS, fixed in buffered formalin andpermeablized in 0.2% triton in PBS. Cells were incubated with indicatedantibodies for 90 min, and if necessary incubated with a specificsecondary antibody, and then mounted in DAPI containing mounting media.Analysis was performed with a confocal microscope (model LC510, CarlZeiss). Proteins examined are as follows: CD-31 (protein present atintercellular junctions that is necessary for cell to cell attachment),Fak (focal adhesion kinase), Paxillin, Vinculin (critical adhesionassembly proteins), WASP (Wiskott Aldrich Syndrome protein, required fornucleation and elongation of F-actin fibers), β-catenin (required forcell survival, regulation of cell surface proteins).

In the CD31/PECAM1 detected cells, pronounced CD31/PECAM localizationwas found at cell/cell junctions in P28 treated cells as compared tocontrol (FIG. 4A). In the paxillin detected cell, the paxillin wasmainly localized on cell surface of the control cells, however it wasmore often found on F-actin fibers in the P28 treated cells (FIG. 4B).In the Fak detected cells, Fak was mainly on localized cell surface ofthe control cells, while it was more often found on F-actin fibers ofthe P28 treated cells (FIG. 4C). In the WASP detected cells, at 4 h WASPlocalization was mostly nuclear in control cells, while WASP waslocalized on the nucleus and at the cell surface in P28 treated cells(FIG. 4D). At 24 h, WASP was mostly localized at the cell surface incontrol cells, while it was mostly localized in the nucleus in P28treated cells (FIG. 4D). In the vinculin detected cells, vinculin waslocalized mainly on the cell surface in control cells, while vinculinwas more often localized on F-actin fibers in P28 treated cells (FIG.4E). In β-catenin detected cells, at 4 h, β-catenin localization wasmostly cytoplasmic with some on the cell surface in the control cells,while β-catenin was mostly localized on the cell membrane with some inthe perinuclear space in the P28 treated cells. At 24 h, β-cateninlocalization was mostly on the cell membrane and in the nucleus in thecontrol cells, while β-catenin was localized on the cell membrane andperinuclear area in P28 treated cells. Therefore, the presence of P28prevented the structural changes normally found in HUVECs undergoingangiogenesis.

Example 5 In vitro Growth Inhibition of Human Melanoma Cells by P28

The ability of P28 to inhibit the growth of human melanoma Mel-2 cellsin vitro was determined. Mel-2 cells were plated in 24 well cultureplates at 10,000-12,000 cells/well and allowed to attach to the plateovernight. Cells were then incubated at 37° C. in media alone (MEM-Ewith 10% FBS) or media containing P28 peptide. P28 was added at 5 μM, 50μM, and 100 μM. The number of cells in each well was counted at Oh, 24h, 48 h and 72 h. The number of cells in each well was counted using aCoulter counter at the indicated time.

The results show that P28 inhibits growth of Mel-2 cells in aconcentration dependent manner. P28 inhibited the Mel-2 cell growth byabout 50% at 100 μM and 24 h (FIG. 5). These results indicate that P28inhibits the growth of cancer cells, specifically human melanoma-2cells.

Example 6 In Vivo Anti-Tumor Activity of P28 Peptides

One million mel-2 cells were injected subcutaneously into the dorsalflank of 3-4 week old athymic mice (n=13 per group). Animals receiveddaily i.p. injections of PBS only, 8 mg, or 16 mg per kg body weight(b.w.) of P28 peptide in PBS. Animals were examined daily for thedevelopment of palpable tumors. Once the tumor developed, tumor size wasmeasured using a caliper and tumor volume was determined.

P28 inhibited the tumor incidence and growth in the mice. With thetreatment of 16 mg/kg b.w., about 50% of the animal were tumor-free 40days after the mel-2 cells were injected, while only about 95% of thecontrol animals had tumors 22 days after the mel-2 cells were injected(FIG. 6A). P28 also inhibited the growth of the tumors by about 30% at20 days post treatment with 16 mg/kg b.w. P28 (FIG. 6B). These resultsindicate that P28 can prevent the slow and prevent the develop oftumors, as well as slow the growth of existing tumors in vivo, and thuswould make an effective therapeutic for cancer prevention and treatmentin humans.

1. An expression vector, which encodes an isolated peptide that iscapable of inhibiting capillary tube formation by HUVEC cells in vitro,wherein the isolated peptide consists of SEQ ID NO: 2 or a truncation ofSEQ ID NO: 2, and wherein the truncation comprises amino acid residues1-18 of SEQ ID NO:
 2. 2. The expression vector of claim 1, wherein saidisolated peptide consists of amino acid residues 1-18 of SEQ ID NO: 2.